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An Extraction of Spinach
The extraction procedure in spinach is very important because it helps isolate the
pigments that will be observed. Extraction exploits solubility and leaves the insoluble molecules
behind. The importance of the blender is to change the spinachs physical properties into a
smaller surface area in order for the other chemical ingredients to mix in much faster. The
soluble components of the blended spinach are soluble in hexane/acetone solution. The reason
why it is neither pure hexane nor acetone is because, acetone is miscible in water, and the
pigments are not adequately soluble in hexane alone. The use of the salt (NaCl) is essential
because the presence of salt in water is forcing the organic components of the spinachs organic
layer due to the reduced solubility.
The main pigments of the spinach are chlorophyll, carotene, xanthophyll, and
pheophytin. Carotene is a pigment whose color, ranges from yellow orange to orange and exists
in two forms, known as - and -, which differ in the position of their double bond in the
outermost cyclohexene rings. The rest of the carotene are consists of methyl groups (

) and
conjugated polyenes (alternating single and double bonds). It is considered the least polar of the
pigments because it doesnt have a more polar functional group unlike the other pigments being
extracted. Chlorophyll is the green pigment found in the plants chloroplasts, which exist in two
structures; chlorophyll a and b. Although both have a structure called porphyrin ring, the
difference between the two is that, one of the methyl groups in the chlorophyll a

is
converted into an aldehyde function in chlorophyll b . Chlorophyll b tend to be
more polar than chlorophyll a. Pheophytin is a grey pigment that is related to chlorophyll, except
that its porphyrin ring doesnt have

ion on its center but two different protons instead. Like

chlorophyll, it has two different structure; pheophytin a

, and pheophytin b .
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Pheophytin a is less polar than pheophytin b (which may not be visible). Due to the differences
in the porphyrin ring central atom, chlorophyll is considered to be more polar than pheophytin.
The last pigment mentioned is the xanthophyll which is a yellow colored pigment and is
considered the most polar of all the colored dyes because it can exhibit van der waals, dipole-
dipole, and Hydrogen-bonding interactions. Like carotenes, it has conjugated polyenes and
methyl groups but, it has alcohol group ( located at it each end.
After doing the column chromatography and TLC, it is revealed that the carotenes has the
highest

value and is therefore considered as the least polar of all the pigments. It is followed
by pheophytin a (because pheophytin b is not visible), then chlorophyll a. Chlorophyll b and
xanthophyll are the two most polar of the pigment dyes, but xanthophyll is the most polar
because it has the lowest

value. The reason behind this is because of their differences in

attraction to stationary phase (which is very polar) and their solubility in the eluents used. As
observed, when the TLC plate is placed on the developing jar, the solvent and solute tend to go
on a race and the more polar pigment will travel a smaller distance on the TLC plate than those
that are not.
The eluent used were hexane, 75/25 hexane/acetone, and acetone. The least polar among
them is hexane and is therefore used first (on carotene extraction) also because carotene is the
least polar and if more polar eluent were used, all the components will be washed down and that
is not the aim of this experiment. It is just logical to use the eluents according to their polarity
ranking, and the order of polarities among the different pigments: hexane/acetone on chlorophyll
and pheopytin, and lastly, acetone on the most polar pigment, xanthophyll.
References
Padias, A. (2013). Making The Connections. (2nd ed.). Plymouth, MI: Hayden-McNeil
Publishing.
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Padias, A. (2013). Organic Chemistry Laboratory Manual. (4th ed., pp. 01-06). Plymouth, MI:
Hayden-McNeil Publishing.
Isopentyl acetate. (n.d.). Retrieved from
http://www.helsinki.fi/kemia/opettaja/aineistot/hyonteistenkemiaa/english/isopentyl
acetate_webpage.htm


















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Student Name: Denisse Cuarteros
Lab # 3: An Extraction of Spinach
Performed: September 18, 2013
T.A: Will Brezinksi
Chem 243A-035